The object of this proposal is to study the regulatory mechanisms of transcription in human cells. Human adenovirus is chosen as the model system. Successful development of an in vitro functional assay for gene activity in my laboratory has made possible the study of the mechanism of interactions of RNA polymerases II and III plus necessary factors with adenovirus genes. This year I have located the control regions surrounded or within the 5.5S RNA gene by using the truncated DNA as templates in this system. The system has been further fractionated. A full activity can be restored by combining the necessary components. In addition to the RNA polymerase III system, three systems of RNA polymerase II were developed from wheat germ, calf thymus and human KB cells. Discrete transcripts were synthesized from restriction fragments of adenovirus DNA. The goals set for the coming year are isolation of factors, further locating the control regions on DNA by in vitro mutagenesis and characterization of RNA polymerase II transcripts.